Oral tolerance is a long held observation that oral antigen results in systemic hypo-responsiveness. Recently, there is increased interest in oral tolerance following the demonstration that orally administered antigen can suppress several animal models of autoimmune disease. In addition, the oral administration of autoantigen is being tested in human autoimmune diseases and in transplantation. Nonetheless, a great deal remains to be learned about the basic mechanisms that underlie immune responses in the gut associated lymphoid tissues (GALT). Effector mechanisms of immune tolerance induced by oral antigen have been defined and relate to dose of protein fed. Low doses of protein favor induction of regulatory T cells that secrete Th2 cytokines and TGF-beta whereas higher doses induce anergy or deletion. The inductive events, however, that govern the generation of oral tolerance are not well understood and investigation of the inductive events in oral tolerance is the focus of this grant application. Based on our preliminary data we hypothesize that multiple factors contribute to the induction of regulatory cells in the GALT that mediate low dose oral tolerance including the unique micro-environment (cytokine milieu) in the gut, specialized antigen presenting cells (most likely B cells) that handle incoming antigen and the predominant expression of B7.2 on GALT antigen presenting cells. We also propose that these factors promote low affinity interactions between peptide-MHC-TCR leading to preferential generation of TGF-beta and Th2 responses. Feeding higher doses results in deletion or anergy in the GALT and at higher doses antigen leaks in the bloodstream and induces systemic tolerance. In our investigations, we will study altered peptides, which vary in their affinity for the TCR or MHC and will provide unique tools to address the issue of peptide-MHC-TCR affinity in the induction of oral tolerance. This together with studying oral tolerance in B-less mice and altering the expression of costimulatory molecules (B7.1 or B7.2) will allow us to define the role antigen, antigen presenting cells and costimulatory molecules in the inductive limb of oral tolerance. Thus the three specific aims of our proposal are: 1) How does the affinity of a peptide for binding to MHC II or the TCR affect the induction of regulatory cells following oral antigen administration? 2) What is the role of B cells in the induction of oral tolerance? and 3) What is the role of costimulation in the induction of oral tolerance? Addressing these questions will provide basic information regarding mechanisms of the induction or oral tolerance and immune responses in the gut associated lymphoid tissue.